The advent of super-resolution imaging (SRI) has created a need for optimized labelling strategies. We present a new method relying on fluorophore-conjugated monomeric streptavidin (mSA) to label membrane proteins carrying a short, enzymatically biotinylated tag, compatible with SRI techniques including uPAINT, STED and dSTORM. We demonstrate efficient and specific labelling of target proteins in confined intercellular and organotypic tissues, with reduced steric hindrance and no crosslinking compared with multivalent probes. We use mSA to decipher the dynamics and nanoscale organization of the synaptic adhesion molecules neurexin-1β, neuroligin-1 (Nlg1) and leucine-rich-repeat transmembrane protein 2 (LRRTM2) in a dual-colour configuration with GFP nanobody, and show that these proteins are diffusionally trapped at synapses where they form apposed trans-synaptic adhesive structures. Furthermore, Nlg1 is dynamic, disperse and sensitive to synaptic stimulation, whereas LRRTM2 is organized in compact and stable nanodomains. Thus, mSA is a versatile tool to image membrane proteins at high resolution in complex live environments, providing novel information about the nano-organization of biological structures.
Study design: Longitudinal, descriptive design. Objectives: The aim of this study was to investigate the frequency, cause and duration of rehospitalisations in individuals with spinal cord injury (SCI) living in the community. Setting: Australian spinal cord injury unit in collaboration with State Health Department. Methods: A data set was created by linking records from the NSW Department of Health Inpatient Statistics Collection between 1989-1990 and 1999-2000 with data from the Royal North Shore Hospital (RNSH) Spinal Cord Injuries Database using probabilistic record linkage techniques. Records excluded were nontraumatic injuries, age o16 years, spinal column injury without neurological deficit, full recovery (ASIA Grade E) and index admission not at RNSH. Descriptive statistics and time to readmission using survival analysis, stratified by ASIA impairment grade, were calculated. Results: Over the 10-year period, 253 persons (58.6%) required one or more spinal-related readmissions, accounting for 977 rehospitalisations and 15,127 bed-days (average length of stay (ALOS) 15.5 days; median 5 days). The most frequent causes for rehospitalisation were genitourinary (24.1% of readmissions), gastrointestinal (11.0%), further rehabilitation (11.0%), skin-related (8.9%), musculoskeletal (8.6%) and psychiatric disorders (6.8%). Pressure sores accounted for only 6.6% of all readmissions, however, contributed a disproportionate number of bed-days (27.9%), with an ALOS of 65.9 (median 49) days and over 50% of readmissions (33 out of 64) occurred in only nine individuals aged under 30 years. Age, level and completeness of neurological impairment, all influenced differential rates of readmission depending on the type of complication. Overall rehospitalisation rates were high in the first 4 years after initial treatment episode, averaging 0.64 readmissions (12.6 bed-days) per person at risk in the first year and fluctuating between 0.52 and 0.61 readmissions (5.1-8.3 bed-days) per person at risk per year between the second to fourth years, before trending downwards to reach 0.35 readmissions (2.0 bed-days) as 10th year approaches. Time to readmission was influenced by degree of impairment, with significantly fewer people readmitted for ASIA D (43.2%) versus ASIA A, B and C (55.2-67.0%) impairments (Po0.0001). The mean duration to first readmission was 46 months overall, however, differed significantly between persons with ASIA A-C impairments (26-36 months) and ASIA D impairment (60 months). Conclusion: Identifying rates, causes and patterns of morbidity is important for future resource allocation and targeting preventative measures. For instance, the late complication of pressure sores in a small subgroup of young males, consuming disproportionately large resources, warrants further research to better understand the complex psychosocial and environmental factors involved and to develop effective countermeasures.
The coupling between the quaternary structure, stability and function of streptavidin makes it difficult to engineer a stable, high affinity monomer for biotechnology applications. For example, the binding pocket of streptavidin tetramer is comprised of residues from multiple subunits, which cannot be replicated in a single domain protein. However, rhizavidin from Rhizobium etli was recently shown to bind biotin with high affinity as a dimer without the hydrophobic tryptophan lid donated by an adjacent subunit. In particular, the binding site of rhizavidin uses residues from a single subunit to interact with bound biotin. We therefore postulated that replacing the binding site residues of streptavidin monomer with corresponding rhizavidin residues would lead to the design of a high affinity monomer useful for biotechnology applications. Here, we report the construction and characterization of a structural monomer, mSA, which combines the streptavidin and rhizavidin sequences to achieve optimized biophysical properties. First, the biotin affinity of mSA (Kd = 2.8 nM) is the highest among nontetrameric streptavidin, allowing sensitive monovalent detection of biotinylated ligands. The monomer also has significantly higher stability (Tm = 59.8°C) and solubility than all other previously engineered monomers to ensure the molecule remains folded and functional during its application. Using fluorescence correlation spectroscopy, we show that mSA binds biotinylated targets as a monomer. We also show that the molecule can be used as a genetic tag to introduce biotin binding capability to a heterologous protein. For example, recombinantly fusing the monomer to a cell surface receptor allows direct labeling and imaging of transfected cells using biotinylated fluorophores. A stable and functional streptavidin monomer, such as mSA, should be a useful reagent for designing novel detection systems based on monovalent biotin interaction. Biotechnol. Bioeng. 2013; 110: 57–67. © 2012 Wiley Periodicals, Inc.
Continuous methadone treatment during pregnancy is associated with earlier antenatal care and improved neonatal outcomes. Innovative techniques for early engagement in methadone treatment by pregnant heroin using women or those planning to become pregnant should be identified and implemented.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.